This invention relates to the field of cancer diagnosis and the application of diagnostic techniques in pathology and hematology. Specifically, the invention relates to techniques that indicate the presence of chromosomal aberrations by detecting tumor-specific gene products that are exclusively expressed by tumor cells containing the chromosomes.
Chromosomal abnormalities or aberrations are a leading cause of genetic disorders or diseases, including congenital disorders and acquired diseases, such as malignancies. Malignant cells have a common clonal origin as they are believed to originate from a single autonomously growing cell that withdrew from environmental growth regulating signals.
The term xe2x80x98cancerxe2x80x99 comprises a heterogeneous group of neoplasms, in which each type has its own characteristic when considering its malignant potential and its response to therapy. Currently, the effectiveness of cancer treatment is empirically determined. Depending on the moment in time in the development of cancer, the origin and spread of the cancer, and on the physiological condition of the patient, the most proper and most effective treatment is selected. At present, selections from surgical treatment, radiation therapy and chemotherapy (or combinations of the former therapies) can be made. Yet, it is realized that each therapy bears side-effects that compromise the benefits of treatment enormously. It goes without saying that accurate diagnosis of the various cancer types is pre-eminent in helping select the most effective therapy.
The basis of cancer stems from chromosomal aberrations such as translocations, inversions, insertions, deletions and other mutations within or among chromosomes. Often, one chromosome or two different chromosomes are involved in the development of malignancies. In this way, genes, or fragments of genes are removed from the normal physiological context of the non-aberrant chromosome and fuse with or find a location in a recipient chromosome, (be it the same or a second chromosome) adjacent to non-related genes or fragments of genes (often oncogenes or proto-oncogenes), where the new genetic combination can be the foundation of a malignancy.
Rearrangements, such as translocations happen often in a somewhat established pattern, where genes, or fragments thereof, are removed from the non-aberrant chromosome at a breakpoint or breakpoint cluster region, and are inserted in the recipient chromosome at a fusion region, thereby creating rearranged, deleted, translocated or fused genes that are specific for that specific cancer. Moreover, rearrangements or translocations can be reciprocal, in that two chromosomes exchange parts which leads to cells containing two, reciprocally rearranged chromosomes which both contain new fused genes.
When the fused gene is translated, it generates a gene-product, mRNA, that is unique for the tumor. The chimeric mRNA comprises parts or fragments of two mRNA""s that correspond to and were originally transcribed by the originally separated genes. This tumor-specific mRNA is uniquely characterized by a fusion point, where the RNA fragments meet. In some cases, these fusion points can be detected by hybridizing nucleic acid probes. However, considering the large variation within the individual rearrangements seen in these translocations and depending on the localization of the breakpoint within the non-aberrant gene wherein (even when the translocations occur within the same two genes) different tumor-specific genes can be generated, it is deemed likely that within each separate case of these types of cancer, new fusion points arise. Detection of cancer by specific detection of the fusion-point of the tumor-specific gene-product (mRNA) has therefore never been widely applicable.
When the fused gene is fused in frame, the fused mRNA is translated into a fusion protein that is unique for the tumor. The protein comprises parts of two proteins that correspond to and were originally transcribed by and translated from the originally separated genes. Tumor-specific proteins are uniquely characterized by a fusion point, where the two proteins meet. Fusion points are antigenically exposed, comprising distinct epitopes which sometimes can be immunologically detected. However, considering the large variation within the individual rearrangements seen in these translocations and depending on the localization of the breakpoint within the non-aberrant gene wherein (even when the translocations occur within the same two genes) different tumor-specific genes can be generated, it is deemed likely that within each separate case of these types of cancer, new fusion points arise. Detection of cancer by specific detection of the fusion-point epitope of the tumor specific protein has therefore never been widely applicable. The tumor-specific gene products (fusion products) of the fused or rearranged genes may contribute to the further development of the cancer.
An area where chromosomal aberrations are relatively well studied (as compared with other cancer types) is the field of leukemia. Comparable to most malignant tumors, leukemias differ in the degree of differentiation of tumor cells. According to clinical presentation, leukemias are divided in to acute and chronic forms, depending on the rapidity with which they evolve and, if untreated, cause death.
Depending on the cell lineage(s) involved in the leukemic process, acute leukemias are classified as acute lymphoblastic leukemias (ALL) and acute non-lymphoblastic leukemias (ANLL), with ALL the most predominant type ( greater than 80%) occurring in childhood. Chronic leukemias are malignancies in which the uncontrolled proliferating leukemic cells are capable of maturation. Two subtypes of chronic leukemia are distinguished, chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Within these four groups, a considerable heterogeneity in biology and prognosis is seen, which currently is stratified along morphological features. This stratification bears, as yet, little value as to an understanding and prediction of the prognosis of a leukemic patient and to rational therapy design.
However, recent molecular genetic studies of leukemic patients have shown that a wide variety of chromosomal aberrations can be found with the various forms of leukemia. One group consists of immunoglobulin (IG) or T-cell receptor (TCR) gene rearrangements, comprising antigen-receptor gene rearrangements that go beyond the normal, physiological processes that are required to generate the diversity of the antigen receptor molecules which typify the lymphoid cell population. In one large group of IG and TCR rearrangements known to be associated with leukemia, tumor specific antigen receptor molecules are expressed. Another group of aberrations comprise deletions of a whole gene or parts of a gene from a genome. As a result of the deletion, promotor regions normally belonging to the now deleted gene can exert control over another gene, resulting in aberrant transcription the gene. An example is the deletion of the coding regions of the SIL gene in T-cells, resulting in the transcription of the normally not expressed TAL-1 gene in T-cells, resulting in ectopic expression of TAL-1 fusion protein. Yet another group comprises translocations of gene fragments between chromosomes, resulting in fusion genes that may well transcribe unique fusion proteins that contribute to the development of the malignancy. Well known examples are the translocations resulting in BCR-ABL fusion genes found in  greater than 95% of cases of CML and in 30% of cases of adult ALL and TEL-AML1 which is found in 25-30% of cases of childhood ALL. However, many more fusion genes, such as E2A-PBX1, ETO-AML1 and PML-RARa are known.
Chromosomal aberrations can be detected by a wide array of techniques, various of which entail modern biomolecular technology. Traditional techniques such as cytogenetic analysis by conventional chromosomal banding techniques are, although highly precise, very labor intensive, require skilled personnel, and are thus expensive. Automated karyotyping is useful for some diagnostic applications, such as prenatal diagnosis, but is ineffective in analyzing complex characteristics of malignancies. Furthermore, it is possible to detect increased activity of proteins, for example tyrosine-kinase activity (see, PCT International Publication WO 95/31545) in tumor specific cells. The foregoing techniques require fresh cells, which are not always available.
Other, more modern, techniques using Southern blotting or other nucleic acid hybridization techniques or amplification techniques such as PCR, for detecting well-established chromosomal aberrations for which suitable nucleic acid probes or primers are available. With these techniques, fresh or frozen cells can be used, and sometimes even older samples which have been stored appropriately (such as after formalin fixation), as long as the nucleic acid to be hybridized or amplified remains accessible and intact. However, even with this modern technology, several disadvantages can be found that hamper the application of these diagnostic techniques in the rapid screening for chromosomal aberrations related to such malignancies.
For instance, Southern blotting takes 3 to 4 weeks, which is much too slow to permit therapeutic intervention in malignancies, and allows only 10-15 kb of nucleic acid to be analyzed per probe analysis.
PCR, although in essence well-suited for rapid and massive diagnostic testing or even screening, allows for the analysis of only 0.1 to 2 kb of nucleic acid (DNA or RNA) per PCR analysis, which greatly hampers the rapid screening of vast stretches of chromosomes and breakpoint cluster or fusion regions within the chromosomes or their gene-products. An additional disadvantage of PCR is its inherent sensitivity to mismatched primers. Small, normal and physiological, alterations which can always be present in the nucleic acid sequence of the gene fragment complementary to the primer will make it impossible to operate the PCR with the wanted effect and may result in misdiagnosis and false-negative results. Especially false-negative results render a PCR-based diagnostic test, albeit very specific, insufficiently sensitive for reliable diagnosis, and it goes without saying that only a reliable diagnosis of malignancies can contribute to an understanding of the prognosis and the design of an adequate therapy.
Fluorescent in situ hybridization techniques (FISH) are not so strongly dependent on the exact matching of nucleic acid sequences to get positive diagnostic results, but can only be employed for the detection of chromosomal DNA and not for the detection of the gene-products of the chromosomes. In general, FISH employs probe analysis with large, mainly unspecified, nucleic acid probes that hybridize, however often with varying stringency, with the genes or gene fragments located in the rearranged chromosome in the malignant cell. Using large probes renders the FISH technique very sensitive. The binding of the probes is detected by subsequent detection of the probes with (often multiple) fluorochromes via microscopic observation of a population of cells obtained from the tested sample.
However, even the currently used FISH protocols have inherent disadvantages, mainly relating to the selection of nucleic acid probes employed in the current FISH protocols, often resulting in false-positive results in the diagnosis of chromosomal aberrations, resulting in diagnostic tests that are, although sensitive, not very specific, at least not specific enough to employ standard FISH techniques in massive or rapid diagnostic testing, let alone in automated testing or screening. A false-positive result necessitates cumbersome re-testing of patients, or even unsuspecting clients that have been submitted to routine screening protocols, and can greatly alarm these people.
Immunological detection of the fusion proteins resulting from chromosomal aberrations has, although widely tried, never been successful. This failure is caused mainly by the fact that it is hard to find immunological reagents that are exclusively reactive with tumor-specific proteins contrary to immunological detection of non-fusion proteins that are normally also produced by the body, albeit at a lower level (see, for example, Nagasaki et al., J. Imm. Methods 162, 235-245, 1993). Usually, such antibodies cross-react with normal cellular proteins. Only when specific fusion points are known, may it be possible to select specific immunological reagents that react exclusively with the tumor-specific protein, by selective binding to the fusion point epitope. However, the variation in fusion points is so large that specific immunological detection only works in a few occasions, often solely on a patient-by-patient basis.
Furthermore, the identified diagnostic tests have the great inherent disadvantage that they require specialized and well equipped laboratories and trained and highly skilled personnel. Furthermore, these tests are only used in suspected cases of malignancies, and are not suitable for large scale screening of populations at risk for the presence of chromosomal aberrations. Large scale and preventive screening may lead to the early detection of malignancies, after which the often fatal course of a malignancy can be intercepted in an early phase of its development.
The present invention now provides a method to be used in diagnostic testing of biological samples such as blood samples, serum samples, samples of cells, tissue samples, bone marrow, biopsies, for chromosomal aberrations. The invention provides a method to be used in diagnostic testing where both a high sensitivity as well as a high specificity is required. The invention provides a method that can optionally be performed in routine laboratories by personnel with ordinary skills.
The present invention is characterized by a method of detecting chromosomal aberrations in a biological sample via the exclusive detection of tumor-specific gene-product using at least two different probes directed against the tumor-specific gene-product originating from the chromosomal aberration. A surprising advantage of the invention is that it provides a method of detecting chromosomal aberrations related to a wide array of types of cancer, for example, the invention provides a method to detect chromosomal aberrations related to leukemia.
The invention provides a method to detect tumor-specific gene products of various types of chromosomal aberrations. For example, the invention provides a method to detect gene-products corresponding to the fused genes found in chromosomal deletions, inversions or translocations. As an example of the invention a method is provided of detecting the Philadelphia chromosomal aberration found in leukemias. The invention provides a method of detecting tumor specific gene-products such as tumor-specific mRNA as well as tumor-specific protein. The probes used by the invention are optionally adjusted to the nature of the gene product, mRNA detection is provided by using at least two different nucleic acid probes, each being reactive with distinct sites on the gene-product. Tumor-specific protein detection is provided by using as probes at least two different binding-proteins, each being reactive with distinct sites on the gene product. As binding proteins, a wide array of proteins is known in the art, such as receptor molecules, polyclonal or monoclonal (synthetic) antibodies, binding peptides or xe2x80x98phagexe2x80x99 antibodies derived via phage display techniques, and so on. By using antibodies, the invention provides a method to detect chromosomal aberrations immunologically.
As an example of the invention, a method is provided wherein the tumor-specific gene-product is detected by a sepharose-Western blotting procedure. As a yet another example of the invention, a method is provided wherein the tumor-specific gene-product is detected by dip-stick assay. However, other methods, wherein the tumor-specific gene product is detected by at least two different probes are also provided by the invention. For example, the invention provides a method wherein mRNA derived from a fused gene is detected by at least two nucleic acid probes, wherein at least one is directed against a mRNA fragment comprising the 5xe2x80x2 site of the tumor-specific mRNA, and at least one other one is directed against a mRNA fragment comprising the 3xe2x80x2 site of the tumor-specific mRNA, said fragments each corresponding to a non-tumor-specific mRNA.
Furthermore, the invention provides a method wherein protein derived from a fused gene is detected by at least two binding proteins, at least one is directed against a protein fragment comprising the amino-terminal fragment of the tumor-specific protein, and at least one other is directed against a protein fragment comprising the carboxy-terminal fragment of the tumor-specific protein, the fragments each corresponding to a non-tumor-specific protein. As an example, the invention provides a method of detecting tumor-specific gene product wherein the amino-terminal protein fragment of the gene product corresponds to the ABL or BCR protein whereas the carboxy-terminal protein fragment corresponds to the BCR or ABL protein, respectively. With this example, probes are used that have similar antigen specificities as seen for antibodies 7C6, ER-FP1, Yae, 8E9, G98-271.1.3, as shown in the experimental part herein more throughly described. Furthermore, the invention provides a method wherein protein derived from a fused gene is detected by flow cytometric detection by at least two binding proteins, wherein at least one is directed against a protein fragment comprising the amino-terminal fragment of the tumor-specific protein, and at least one other one is directed against a protein fragment comprising the carboxy-terminal fragment of the tumor-specific protein, said fragments each corresponding to a non-tumor-specific protein. As an example, the herein described bead-based sandwich antibody technique allows easy and rapid flow cytometric detection of different types of fusion proteins, preferably in a single tube assay, by using different bead-bound catching antibodies against one part of the different fusion proteins and the relevant corresponding detection antibodies against the other part of the fusion proteins.
For reasons of efficacy, it is preferred to investigate the occurrence of different fusion gene proteins simultaneously in one tube. This preference is not because a particular malignancy will have more than one fusion gene protein, but because it is convenient to have a single test tube for detection of several well-established fusion gene proteins within one disease category. Based on different flow cytometric characteristics of the beads (e.g., size, fluorochrome color, intensity of fluorochrome staining, or side scatter characteristics), multiple fusion proteins can be specifically detected in the same assay. This also includes the detection of fusion proteins from various variant translocations of the same target gene as well as fusion proteins from translocations with variant breakpoints. Of course, the herein described flow cytometric detection method can also be applied to fusion gene products of a nucleic acid nature, wherein different nucleic acid probes are labeled with different beads or fluorochromes as described herein.
The invention provides a method using probes that can be labeled or conjugated with reporter molecules, such as biotin, digyoxigenin, enzymes such as peroxidase, alkaline phosphatase, or other reporter molecules or reporter particles, such as beads, known in the art. The invention further provides a diagnostic kit comprising all the means, such as (labeled) probes or reagents or substrate or instructions, necessary to carry out the method according to the invention. Methods or diagnostic kits provided by the invention are preferably used to detect chromosomal aberrations found with certain types of cancer, for example with leukemia, be it in the detection of (residual) cancer in patients or the screening for cancer in larger populations as a whole.